JP7465014B2 - Composition for hair growth and/or hair care - Google Patents

Description

The present invention relates to a composition that promotes hair growth and/or hair development.

Plasmalogen is a type of phospholipid with antioxidant properties, and is one of the glycerophospholipids. Plasmalogen is present in all mammalian tissues, accounting for approximately 18% of the phospholipids in the human body, but is known to be particularly abundant in the brain, nerves, cardiac muscle, skeletal muscle, white blood cells, and sperm.

Plasmalogen is known to promote neurogenesis, inhibit neuroinflammation caused by lipopolysaccharide (LPS), and inhibit the accumulation of amyloid beta (Aβ) protein in the brain, and is said to be effective in treating neurological diseases such as Alzheimer's disease, Parkinson's disease, depression, and schizophrenia. For example, Non-Patent Document 1 reports that memory function in mild Alzheimer's disease improved in patients who were orally administered purified plasmalogen derived from scallops.

Meanwhile, in recent years, thinning hair has become a serious problem not only for men but also for women. Hair consists of a hair shaft that protrudes from the scalp and a hair root inside the scalp, and the hair bulb at the base of the hair root contains hair matrix cells that produce hair and hair papilla cells that control the function of hair matrix cells. This hair root is fixed to the scalp by a hair root sheath consisting of an inner root sheath and an outer root sheath. The outer root sheath has a bulge region where hair root stem cells and melanocyte stem cells exist, and also contains "CD34-positive cells" that are the source of hair matrix cells, so it is thought to be one of the essential elements for hair production.

It is also believed that promoting the proliferation of hair matrix cells, which produce hair shafts, and hair root sheath cells around the hair roots is important in eliminating thinning hair.

Furthermore, it is known that an increase in new blood vessels in the scalp can efficiently provide nutrients to the hair roots, promoting hair growth and/or hair development.

Given the circumstances described above, little research has been conducted to date on the effects of plasmalogens on hair growth and/or hair development.

Fujino T.et al, “Efficacy and Blood Plasmalogen Changes by Oral Administration of Plasmalogen in Patients with Mild Alzheimer's Disease and Mild Cognitive Impairment: A Multicenter, Randomized, Double-blind, Placebo-controlled Trial” EBioMedicine, [17] (2017) 199-205

The object of the present invention is to provide a composition having excellent hair growth and/or hair development effects.

As a result of intensive research conducted by the inventors to solve the above problems, they discovered that plasmalogen promotes phosphorylation of AMPK (AMP-activated protein kinase) in adult human hair follicle outer root sheath cells and promotes expression of vascular endothelial growth factor (VEGF) in human fibroblasts, thereby exerting an excellent effect on hair growth and/or hair development, and thus completed the present invention.

That is, the present invention is as follows.
[1] A composition for hair growth and/or hair care, comprising a plasmalogen.
[2] The composition for hair growth and/or hair care described in [1] above, characterized in that the plasmalogen is a plasmalogen extracted from animal tissue.
[3] The composition for hair growth and/or hair care according to [2] above, characterized in that the animal tissue is tissue of an animal selected from the group consisting of shellfish, sea squirts and birds.
[4] The composition for hair growth and/or hair care described in [2] or [3] above, characterized in that the animal tissue is scallop tissue.
[5] The composition for hair growth and/or hair care according to any one of [1] to [4] above, characterized in that the plasmalogen is an ethanolamine-type plasmalogen.

The composition of the present invention has excellent hair growth and/or hair care effects.

FIG. 1 shows the results of promoting AMPK phosphorylation (relative amount of phospho-AMPK to AMPK) in HHORSC cells (human hair follicle outer root sheath cells) by treatment with scallop-derived plasmalogen (0.5 μg/mL, 3 hours). FIG. 1 shows the results of promoting AMPK phosphorylation (relative amount of phospho-AMPK to AMPK) in HHORSC cells (human hair follicle outer root sheath cells) by treatment with scallop-derived plasmalogen (5 μg/mL, 7 hours). FIG. 1 shows the results of VEGF expression in HDF-a cells (human fibroblasts) following treatment with scallop-derived plasmalogen (5 μg/mL, 7 hours). FIG. 1 shows the results of VEGF expression in HDF-a cells (human fibroblasts) following treatment with plasmalogens (5 μg/mL, 7 hours) derived from various animals. Photographs showing the results of hair growth at shaved areas of mice to which a plasmalogen solution was applied (representative examples taken 1 week and 2 weeks after shaving). This is a micrograph of hair roots stained with hematoxylin and eosin from mouse skin to which plasmalogen was applied for four weeks.

The composition for hair growth and/or hair care of the present invention is characterized by containing a plasmalogen.
The composition of the present invention promotes AMPK phosphorylation in human hair follicle outer root sheath cells and VEGF expression in human fibroblasts, and has excellent effects on hair growth and/or hair development. Outer root sheath cells play an important role in hair growth and/or hair development, and activating them can promote hair growth and/or hair development. In addition, VEGF secreted by scalp fibroblasts acts on vascular endothelial cells to increase new blood vessels, which efficiently nourishes the hair roots and promotes hair growth and/or hair development.

That is, the composition containing the plasmalogen of the present invention can be used as a composition for activating AMPK in outer root sheath cells, a composition for promoting VEGF expression, a composition for promoting angiogenesis, and a composition for hair growth and/or hair development. Here, hair growth in the present invention refers to growing lost hair, such as in the treatment of patients with alopecia, and hair development refers to growing existing hair into strong hair, such as in the prevention of thinning hair and hair loss.

The plasmalogen used in the present invention is a type of phospholipid with antioxidant properties, and is one of the glycerophospholipids. It is a subclass unique to glycerophospholipids, characterized by having a vinyl ether bond at the sn-1 position of the glycerol backbone, and has been confirmed in high concentrations in the cell membranes of many mammalian tissues. As a plasmalogen, one having a fatty acid ester bond at the sn-2 position is preferred.

The plasmalogen used in the present invention is not particularly limited as long as it is generally classified as a plasmalogen, but examples thereof include choline-type plasmalogens, ethanolamine-type plasmalogens, inositol-type plasmalogens, and serine-type plasmalogens. Among these, choline-type plasmalogens and ethanolamine-type plasmalogens are preferred, and ethanolamine-type plasmalogens are particularly preferred.

The plasmalogen of the present invention can be extracted from animal tissue. There are no particular limitations on the animal tissue as long as it contains plasmalogen, and examples include aquatic animals such as shellfish, sea squirts, sea cucumbers, salmon, pacific saury, and bonito, and birds. Among these, shellfish, sea squirts, and birds are preferred, and shellfish are particularly preferred. Edible parts (edible parts) are preferred as the parts to be used. These animal tissues may be cut, but it is preferable to use pulverized parts because this allows for more efficient extraction of plasmalogen.

Examples of shellfish include edible bivalve mollusks and snail shells such as scallops, mussels, and abalone, with scallops being particularly preferred. Scallops are edible bivalve mollusks belonging to the Pectenidae family, such as those belonging to the Mizuhopecten and Pecten genera. Specific examples include scallops (scientific name: Mizuhopecten yessoensis) harvested in Japan and European scallops (scientific name: Pectenmaximus (Linnaeus)) harvested in Europe. Edible parts include the adductor muscle and string.

Ascidians are edible chordates belonging to the family Halocynthia, including the genera Halocynthia and Halocynthia. Specifically, Halocynthia roretzi (scientific name) and Halocynthia aurantium (scientific name) can be mentioned. The edible part can be the flesh (fascia).

The bird is not particularly limited as long as it is an edible bird, and examples include chickens, silkie chickens, ducks, etc. As the edible part, breast meat, which is rich in plasmalogens, is preferable.

Extraction of plasmalogen can be performed using water, an organic solvent, or a water-containing organic solvent, and is preferably performed in combination with an enzyme treatment. For example, ethanol extraction and hexane extraction methods can be used, with ethanol extraction being preferred.

The ethanol extraction method is not particularly limited as long as it is a method of extraction using ethanol (including hydrous ethanol), and examples of the method include those described in JP 2019-140919 A, JP 2018-130130 A, JP 2012-039472 A, JP 2010-065167 A, and JP 2010-063406 A.

There are no particular limitations on the hexane extraction method, so long as it is a method of extraction using hexane, and examples of the method include those described in Publication Nos. 2009-154309 and 2008-146942.

The composition of the present invention can be used as an oral or parenteral agent.
Examples of parenteral use include topical preparations and injections. There are no particular limitations on topical preparations as long as they can be applied to the scalp, and examples of the form of topical preparations include ointments, creams, gels, lotions, emulsions, packs, and poultices. Specific examples of topical preparations include hair tonics, shampoos, rinses, pomades, hair lotions, hair creams, hair treatments, and other products that can be used for hair growth and/or hair development.

When used as an oral preparation, the form may be, for example, a tablet, capsule, powder, granule, liquid, grain, rod, plate, block, solid, round, paste, cream, caplet, gel, chewable, stick, etc. Of these, the capsule form is preferred.

The hair growth and/or hair care composition of the present invention is not particularly limited as long as it contains plasmalogen and can be distinguished from other products in terms of its use for hair growth and/or hair care, and examples of such products include so-called health foods, such as pharmaceuticals (including quasi-drugs), cosmetics, and functional foods whose efficacy has been approved by a designated organization, such as foods for specified health uses, foods with nutritional functions, and foods with functional claims. In addition, the scope of the present invention includes products that display on the main body, packaging, instructions, or promotional materials of the product according to the present invention that have a hair growth and/or hair care effect.

For example, in the case of cosmetics or health foods, specific claims such as "promotes hair growth," "promotes hair growth," "prevents thinning hair," "for those concerned about thinning hair," and "for those concerned about hair loss" could be displayed.

The content of plasmalogen in the composition of the present invention may be appropriately determined within the range in which the effect is exhibited. Depending on the form, for example, the plasmalogen is preferably ^10-10 % by mass or more, more preferably ^10-5 % by mass or more, even more preferably 0.1% by mass or more, and particularly preferably 1.0% by mass or more, of the total composition of the present invention, calculated as a dry mass.

When the composition of the present invention is an oral preparation, the amount of intake is not particularly limited, but from the viewpoint of more significantly exerting the effects of the present invention, the plasmalogen intake is preferably 10 ⁻⁶ μg/day or more per adult, more preferably 1 μg/day or more, even more preferably 500 μg/day or more, and particularly preferably 1000 μg/day or more. The upper limit is, for example, 20,000 μg/day, preferably 10,000 μg/day.

The composition of the present invention can be stored as a daily dose in a single container or divided into multiple containers, for example 2 to 3, so that the daily intake amount is the above-mentioned amount.

The composition of the present invention can be produced by known formulation methods by adding ingredients other than the active ingredient (plasmalogen) that are acceptable for oral administration, topical administration or injection, as necessary.

Examples of ingredients other than the active ingredient of the present invention include vitamins, minerals, proteins, peptides, amino acids, animal oils, and vegetable oils. In addition, various ingredients may be blended according to various purposes, such as hydrocarbons, waxes, fats and oils, esters, higher fatty acids, higher alcohols, surfactants, fragrances, dyes, preservatives, antioxidants, UV protection agents, alcohols, and pH adjusters, which are used in hair growth and/or hair restorers.

The present invention will now be described in detail with reference to examples.

We confirmed the effect of plasmalogen, an active ingredient in the composition of the present invention, in promoting AMPK phosphorylation in HHORSC cells (human hair follicle outer root sheath cells).

[Plasmalogen]
The plasmalogen used was ethanolamine-type plasmalogen (PlsEtn) extracted from scallops (scientific name: Mizuhopecten yessoensis) with ethanol and purified by HPLC.

[Cell culture]
Human Hair Outer Root Sheath (HHORSC) cells (#2420) purchased from ScienCell Research Laboratories were used as human hair follicle outer root sheath cells. HHORSC cells were cultured in Mesenchymal Stem Cell Medium (MSCM, #7501). Cells that had been passaged up to 10 times were used in the experiment.

HHORSC cells cultured the previous day were cultured for the specified time (3 or 7 hours) in a mixed medium of 900 μL of FM and a PlsEtn solution in which PlsEtn (0.5 μg or 5 μg) was suspended in 100 μL of Opti-MEM ^TM Reduced Serum Medium (ThemoFisher, #22600050) by sonication.

[AMPK phosphorylation analysis]
HHORSC cells were harvested in buffer A (0.25 M sucrose, 10 mM Hepes-KOH, pH 7.5, 1 mM EDTA, protease inhibitor cocktail) and centrifuged. The cells were suspended in buffer A, disrupted by sonication, and protein was quantified, followed by electrophoresis. The cells were then transferred to a PVDF membrane and detected by Western blotting using Phospho-AMPKα (Thr172) antibody (Cell Signaling Technology, #2535S) and AMPKα antibody (Cell Signaling Technology, #58315). Signals were quantified using Multi Gauge software version 3.0 software (Fuji Film). Signals obtained with Phospho-AMPKα (Thr172) antibody were normalized by dividing the signal obtained with AMPKα antibody. Furthermore, the signal intensity in the cells treated with each treatment was expressed as a relative value, with the value obtained from untreated cells set to 1. Trials were performed more than three times, and the average and standard deviation were shown.

Figure 1 shows the results of AMPK phosphorylation promotion effect (relative amount of phospho-AMPK to AMPK) in HHORSC cells cultured for 3 hours in the presence of 0.5 μg/ml PlsEtn. Figure 2 shows the results of AMPK phosphorylation promotion effect (relative amount of phospho-AMPK to AMPK) in HHORSC cells cultured for 7 hours in the presence of 5 μg/ml PlsEtn.

As shown in Figures 1 and 2, phosphorylation of AMPKα, which is essential for AMPK activation, was enhanced in HHORSC cells cultured in the presence of PlsEtn, compared with cells cultured in the absence of PlsEtn.
From the above results, it is considered that PlsEtn can promote phosphorylation of AMPK in HHORSC cells and promote hair growth and/or hair development.

We confirmed the effect of plasmalogen, an active ingredient in the composition of the present invention, in promoting VEGF expression in HDF-a cells (human fibroblasts).

[Plasmalogen]
As the plasmalogen, ethanolamine-type plasmalogen (PlsEtn) extracted and purified in the same manner as in Example 1 was used.

[Cell culture]
Human dermal fibroblasts-adult (HDF-a) cells (#2320) were used as human fibroblasts. HDF-a cells were cultured in Fibroblast Medium (FM, #2301). Cells up to passage 6 were used in the experiment.

HDF-a cells cultured the previous day were cultured in the presence of 5 μg/ml PlsEtn for 7 hours.
For comparison, HDF-a cells were similarly cultured in the absence of PlsEtn.

[VEGF expression promotion analysis]
Cultured HDF-a cells were harvested in buffer A (0.25 M sucrose, 10 mM Hepes-KOH, pH 7.5, 1 mM EDTA) and centrifuged. The cells were suspended in buffer A, disrupted by sonication, and protein was quantified and electrophoresed. The protein was then transferred to a PVDF membrane and detected by Western blotting using anti-vascular endothelial growth factor (VEGF) antibody (Protein tech #19003-1-AP) and anti-actin antibody (MBL #M177-3). The signal of each protein obtained was quantified using Multi Gauge software version 3.0 software (Fuji Film) and normalized by dividing the VEGF signal by the actin signal. Furthermore, the relative value of the VEGF signal intensity obtained after each treatment was expressed as the average of two trials and the difference between the average, with the untreated value being set to 1.

Figure 3 shows the results of enhanced VEGF expression (relative amount of VEGF to actin) in cultured HDF-a cells.

As shown in Figure 3, VEGF expression was enhanced in HDF-a cells cultured in the presence of PlsEtn compared to cells cultured in the absence of PlsEtn. Therefore, it is believed that PlsEtn can promote the biosynthesis of VEGF, promote angiogenesis, and promote hair growth and/or hair development.

As in Example 2, the effect of plasmalogen in promoting VEGF expression in HDF-a cells (human fibroblasts) was confirmed. In this example, plasmalogens derived from scallops, chicken (scientific name: Gallus gallus domesticus) breast meat, sea squirt (scientific name: Halocynthia roretzi), and mussel (scientific name: Mytilus Linnaeus) were used.

Figure 4 shows the results of enhanced VEGF expression (relative amount of VEGF to GAPDH) in HDF-a cells cultured in the presence of plasmalogens derived from various animals.

As shown in Figure 4, VEGF expression was enhanced by plasmalogens derived from chicken (breast meat), sea squirt, and mussel, as well as scallop-derived plasmalogens. Therefore, it is believed that plasmalogens derived from various animals can promote the biosynthesis of VEGF, promote angiogenesis, and promote hair growth and/or hair development.

The hair growth promoting effect of plasmalogen was confirmed using mice.

[Plasmalogen solution]
A 1% plasmalogen solution was prepared by dissolving 51 mg of scallop-derived PlsEtn and 5 mg of chicken breast-derived ether phospholipid (PL content 9.1%, PlsEtn:PlsCho = 3:4) in 5.2 ml of 70% ethanol.

[Experimental animals]
The experimental animals used were male C3H mice (7 weeks old), which are often used to evaluate hair growth promotion.

[Application of plasmalogen solution to mice]
Ten male C3H mice aged 7 weeks were bred in advance, and at 8 weeks of age, the hair on the dorsal skin was shaved with an electric animal clipper and removed with hair removal cream. 48 hours after shaving, application of a 70% EtOH solution containing 10 mg/ml Pls was started. The application frequency was 5 times/week for 2 weeks, and the application amount was 20 μl/ ^cm2 . As a control, a 70% EtOH solution without plasmalogen was applied at the same frequency and amount. Five mice were used for each treatment group.

[Confirmation of hair growth in mice]
FIG. 5 shows the results of observing the shaved areas of the mice one week and two weeks after shaving.

As shown in Figure 5, two weeks after shaving, the shaved area of the mouse to which the solution containing no plasmalogen was applied had skin with no hair growth in parts, whereas the shaved area of the mouse to which the solution containing plasmalogen was applied had hair growth overall, showing a clear difference in the degree of hair growth. In other words, it was clear that plasmalogen has the effect of promoting hair growth.

It is known that the number of hair root cells increases and the hair roots become larger during the growth phase compared to the resting phase. Therefore, we examined the condition of hair roots in the skin of mice to which plasmalogen had been applied for a certain period of time.

After applying 70% EtOH solution containing 10 mg/ml Pls to mice for 4 weeks in the same manner as in Example 4, mouse skin samples were taken from the applied area and stained with hematoxylin and eosin. As a control, 70% EtOH solution without plasmalogen was applied to mice at the same frequency and in the same amount.

Figure 6 shows a micrograph of hair roots stained with hematoxylin and eosin from mouse skin to which plasmalogen had been applied for four weeks. The scale bar in the figure indicates 50 μm.

As shown in Figure 6, in mouse skin to which a solution containing plasmalogen was applied, a large population of hematoxylin-positive cells, which stains cell nuclei, was observed compared to the control, suggesting that many hair roots were in the growth phase. Therefore, it is believed that plasmalogen can promote increased cell proliferation in hair roots and promote hair growth and/or hair development.

[Formulation Example 1]
A hair growth agent (100 g) was produced according to the formulation shown below.
Scallop extract plasmalogen 0.5mg
Glycerin 0.5mg
Purified water Remainder

[Formulation Example 2]
Hard capsules were produced according to the following formulation.
Scallop extract plasmalogen 0.5mg
Cyclodextrin 3.3mg
Amino acids 1.2mg
Pine index 185.0 mg

The composition of the present invention can be used as an external preparation or an oral preparation and is industrially useful.

Claims (1)

A composition for hair growth and/or hair care, comprising an ethanolamine-type plasmalogen , and being an external preparation or an injectable preparation .

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